"Previous studies in our laboratory have demonstrated that both CD8+ and CD4+ T cells, derived from infiltrates in melanomas, colon carcinomas, breast carcinomas, and lymphomas, can manifest MHC-restricted recognition of tumor-associated antigens (Ag). Identification of MHC class I-restricted Ag recognized by CD8+ cytolytic T cells has led to the development of experimental vaccines for melanoma and other types of cancers. Current projects in our laboratory focus on identifying melanoma associated proteins recognized by CD4+ helper T cells, as well as extending this work to prostate cancer. Tyrosinase, an enzyme expressed by cells of the melanocytic lineage which can be recognized by CD8+ T cells, was also discovered to stimulate CD4+ tumor-infiltrating lymphocytes (TIL) from 2 of 9 melanoma patients examined, and vaccination against tyrosinase is currently under development as an experimental therapy for melanoma. 1) SEARCH FOR TUMOR-ASSOCIATED PROTEINS RECOGNIZED BY CD4+ T CELLS. The identification of Ag recognized by helper T cells is critical to the development of more effective cancer vaccines, which will be designed to recruit multiple arms of the antitumor immune response. In efforts to develop an efficient method for cloning such Ag, a number of strategies are being pursued. Using a biochemical purification approach, a mutated glycolytic enzyme, triosephosphate isomerase, was identified as a unique tumor Ag in one patient's melanoma. The effects of this mutation on enzyme function and possibly tumorigenesis will be investigated. A molecular cloning strategy is also under development, in which cDNA libraries from tumor cells will be expressed as polyhistidine fusion proteins in bacteria, for binding to microscopic beads and uptake and processing by antigen presenting cells. A third approach to Ag identification, involving elution of immunogenic peptides directly from MHC class II molecules on the surface of melanoma cells followed by mass spectrometric sequencing, proved unsuccessful due to limiting amounts of Ag recovered by this technique. 2) DEFINING IMMUNE RESPONSES AGAINST PROSTATE CANCER. Limited information is available on the human immune response to prostate cancer, in part due to a scarcity of cultured prostate cancer lines for testing. We have developed an innovative method for generating immortal cultures from human prostatic epithelium which has proved uniformly successful in establishing over 20 different cell lines from benign and malignant prostatic tissue. Loss of allelic heterozygosity on chromosome 8p, the potential site of a suppresser gene related to prostate cancer, has been used to characterize these lines. Intensive efforts to use these lines as in vitro stimulants to raise tumor-reactive T cells from prostate cancer patients led to the identification of a CD8+ T cell clone recognizing a broadly-expressed prostate cancer-associated Ag. This T cell clone appears to be restricted by a non-polymorphic MHC-like molecule, and further characterization of the Ag and recognition mechanism is in progress. Of note, among 4 prostate cancer patients tested so far, cancer-specific T cells restricted by classical MHC molecules (class I/II) have not yet been identified."